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Am J Cancer Res 2016;6(10):2235-2251
www.ajcr.us /ISSN:2156-6976/ajcr0040144
Original Article
Differences in MEK inhibitor efficacy in molecularly
characterized low-grade serous ovarian cancer cell lines
Marta Llauradó Fernández1, Gabriel E DiMattia2,3,4,5, Amy Dawson6, Sylvia Bamford1, Shawn Anderson7,
Bryan T Hennessy8, Michael S Anglesio1,9, Trevor G Shepherd2,4,5,10, Clara Salamanca9, Josh Hoenisch1,
Anna Tinker11, David G Huntsman1,9,12, Mark S Carey1,13
Departments of 1Obstetrics & Gynaecology, 6Reproductive and Developmental Sciences, 9Pathology and
Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada; 2Translational Ovarian
Cancer Research Program, London Regional Cancer Program, London, Ontario, Canada; Departments of
3
Biochemistry, 4Obstetrics & Gynecology, 5Oncology, 10Anatomy and Cell Biology, University of Western Ontario,
London, Ontario, Canada; 7Department of Urologic Sciences, Vancouver Prostate Centre, University of British
Columbia, Vancouver, British Columbia, Canada; 8Beaumont Hospital and Royal College of Surgeons of Ireland,
Dublin, Ireland; Departments of 11Medical Oncology, 12Molecular Oncology, 13Surgical Oncology, BC Cancer
Agency, Vancouver, British Columbia, Canada
Received September 17, 2016; Accepted September 20, 2016; Epub October 1, 2016; Published October 15,
2016
Abstract: Advanced or recurrent low-grade serous ovarian cancers (LGSC) are resistant to conventional systemic
treatments. LGSC carry mutations in RAS or RAF, leading to several clinical trials evaluating MEK inhibitors (MEKi).
As LGSC cell lines and xenografts have been difficult to establish, little is known about the efficacy and on-target
activity of MEKi treatment in this disease. We compared four different MEKi (trametinib, selumetinib, binimetinib
and refametinib) in novel LGSC patient-derived cell lines. Molecular characterization of these cells included copynumber variation and hotspot mutational analysis. Proliferation, apoptosis and cell viability assays were used to
study drug efficacy. MEKi on-target efficacy was measured using western blotting and isoelectric point focusing
for ERK1/2 phosphorylation. Ten LGSC cell lines were derived from 7 patients with advanced/recurrent disease.
Copy number variation showed significant heterogeneity among cell lines, however all samples showed deletions
in chromosome 9p21.3, and frequent copy number gains in chromosomes 12 and 20. Mutations in KRAS/NRAS
were identified in 4 patients (57%) and RAS mutation status was not associated with higher baseline levels of ERK
phosphorylation. Different degrees of MEKi sensitivity were observed in the LGSC cell lines. Two cell lines, both
with KRAS mutations, were highly sensitive to MEKi. Drug anti-proliferative efficacy correlated with the degree of
inhibition of ERK phosphorylation, with trametinib being the most potent agent. Differences in MEKi efficacy were
observed in LGSC cell lines. Trametinib showed the greatest anti-proliferative effects. This study serves as a basis
for much needed future research on MEKi drug efficacy in LGSC.
Keywords: Low-grade serous ovarian cancer, cell line models, gene mutations, copy-number alterations, MAPK
signalling, MEK inhibitors, ERK1/2 phosphorylation
Introduction
LGSC is much less common than its high-grade
serous counterpart (HGSC), however the 10year survivals are similar for advanced/recurrent cases [1, 2]. LGSC is often diagnosed in
pre-menopausal women and the malignancy
has distinct clinical, pathological and molecular
features compared to HGSC [3-5]. As ovarian
cancer is a heterogeneous disease, it is important that the various molecular subtypes are
studied as distinct diseases [6]. In cases of
advanced and/or recurrent disease, tumor
growth and progression is more indolent than
HGSC, however the disease usually proves to
be fatal [2]. LGSC responds poorly to anti-hormone therapy (9%) or chemotherapy, with
response rates of less than 5% with chemotherapy treatment in the neoadjuvant or relapsed
setting [7-12].
The clinical precursors that lead to the development of LGSC have been characterized. Serous
ovarian borderline tumors (SBOT) are known
MEK inhibition in low-grade serous cell lines
precursor lesions. Certain pathological features associated with SBOT, such as the micropapillary variant (MPSBT), or the presence of
invasive implants are risk factors for developing
LGSC [13, 14]. Alternatively, some cases arise
as primary LGSC presenting with either localized or metastatic disease. Molecular characterization of SBOTs, MPSBT and LGSC [15, 16]
has established a linkage between LGSC and
its precursors, resulting in the pathological
classification of LGSC as a distinct entity. Whole
exome sequencing of LGSC has been reported
and shown that these tumors contain few point
mutations when compared to HGSC [17].
(Vancouver General Hospital and the British
Columbia Cancer Agency (BCCA); London
Regional Cancer Program, London, Ontario,
Canada). Tumor bank protocols, cell line derivation, and the research relating to this study,
was conducted according to institutional
human ethics review board approvals at the
BCCA and the University of British Columbia
(H14-02859 and R05-0119), and Western
University (HSREB 12668E). Clinical information was extracted retrospectively from patient
medical records. Tumor bank pathology was
reported by certified gynecological pathologists to ensure diagnostic accuracy.
The Gynecologic Cancer InterGroup (GCIG)
Consensus Conference recently affirmed an
urgent need to develop specific treatment
options for patients with advanced/recurrent
LGSC [18, 19]. Investigational studies using
MEKi in LGSC are ongoing and previously predicated on experimental evidence showing activated MAPK signalling as a consequence of
either BRAF or RAS mutations, often found in
LGSC [20]. Activating mutations in RAS genes
are one of the most common mutations found
in cancer and multiple allosteric MEK inhibitors
(MEKi), have been developed and used in many
cancer types [21, 22]. Recently, an important
phase II clinical trial evaluated the MEKi selumetinib, reporting response rates of 15% in
patients with recurrent LGSC [23]. These
results compare favourably with a previous
cohort study that reported response rates of
4% using conventional chemotherapy [12].
Several MEKi are now being tested in clinical
trials in patients with advanced/recurrent LGSC
[NCT00551070, NCT01849874, NCT02101788]. Remarkably, preclinical testing of MEKi in
LGSC has been limited due to the absence of
representative cell line and xenograft models
[24]. Thus, the aim of our study was to establish
and molecularly characterize novel LGSC cell
lines derived from patients with advanced/
recurrent disease in order to compare the efficacy of four different MEKi agents (trametinib,
selumetinib, binimetinib and refametinib).
Establishment and maintenance of patientderived LGSC cell lines
Materials and methods
Patients, tumor samples and clinical information
Advanced or recurrent LGSC samples (tumor
and ascites) were obtained from tumor banks
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LGSC patient-derived cell lines were established in-house through continuous in vitro culture of primary patient material (tumor tissue or
ascites) obtained through the OvCaRe Tumor
bank or the London Translational Ovarian
Cancer Research Program (iOvCa241 and
iOvCa250). LGSC cells were established and
maintained in M199:MCDB105 (1:1) media
(Cat. No. M5017, Cat. No. M6395, SigmaAldrich, Oakville, Ontario, Canada) supplemented with 10% fetal bovine serum (dFBS; Cat. No.
SH30070.03, Hyclone, GE Life Sciences,
Logan, UT, USA) at 37°C and 5% CO2. No immortalization methods were used.
Cell line authentication
Microsatellite Analysis of Short Tandem
Repeats (STRs) was performed for cell line
authentication. STR analyses of 10 markers/
loci were performed by Genewiz Inc. (South
Plainfield, NJ) (Supplementary Table 1).
Mutation testing and copy number analysis
DNA was extracted from all cell lines using All
Prep DNA/RNA Mini kit (Cat. No. 80204,
Qiagen, Toronto, ON, Canada) according to protocol instructions, and quantified using a
NanoDrop 2000TM UV-Vis instrument (ThermoScientific, Burlington, ON, Canada). Sequencing
libraries were created from cell DNA for molecular characterization using Ion Torrent
AmpliSeqTM Cancer Hotspot Panel Version 2
(Life Technologies, Grand Island, NY, USA) as
per manufacturer’s protocols. A total of 50
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MEK inhibition in low-grade serous cell lines
Table 1. Clinical information LGSC patients and samples
Age at
Diagnosis
Primary Pathology
Patient #1
58
SBOT with non-invasive implants
IIB
Patient #2
51
SBOT with LGSC
IIIA
Patient #3
53
SBOT with LGSC
IIC
Patient #4
62
Micropapillary SBOT with invasive implants
IIIC
Patient Number
Patient #5
Patient #6
Patient #7
42
42
61
Micropapillary SBOT
LGSC
Primary Peritoneal LGSC
Disease Sample
Stage Number
IC
IIIC
IIIB
Cell Line
Pathology at Collection
Treatment Status
Sample Origin
Mutation Status
1
VOA-1312
2
iOvCa241
LGSC
Naïve
Ascites
KRAS
LGSC
Post-chemotherapy
Ascites
3
VOA-3667*
KRAS
LGSC
Naïve
Tumor Tissue
KRAS, KDR
4
VOA-1056
LGSC
Naïve
Tumor Tissue
NRAS, FGFR3, JAK3
5
VOA-3993
Recurrent LGSC
Post-chemotherapy
Tumor Tissue
NRAS, FGFR3, JAK3
6
VOA-3448
Recurrent LGSC
Post-chemotherapy
Tumor Tissue
KDR, MET, PIK3CA
7
VOA-3723
Recurrent LGSC
Post-chemotherapy
Tumor Tissue
KDR, MET, PIK3CA
8
VOA-4627
Recurrent LGSC
Post-chemotherapy
Ascites
KIT, PIK3CA, TP53
9
VOA-4698
Recurrent LGSC
Post-chemotherapy
Ascites
KIT, PIK3CA, TP53
10
iOvCa250
LGSC
Naïve
Ascites
APC, KDR, KIT
Note: SBOT = Serous Borderline Ovarian Tumor. (*) = Primary cell culture.
Table 2. Missense mutations identified in LGSC cell lines using Ion AmpliSeq Cancer Hotspot Panel v2 Analysis
Gene Name
Mutation ID
APC (Chr.5)
COSM19099
FGFR3 (Chr.4)
COSM1539830
JAK3* (Chr.19)
COSM34213
KDR (Chr.4)
COSM149673
KIT (Chr.4)
COSM28026
KRAS* (Chr.12)
MET (Chr.7)
PATIENT 1
PATIENT 2
PATIENT 3
VOA-1312
iOvCa241
VOA-3667#
COSM520, COSM521 c.35G>T (Ht) c.35G>A (Ht)
PATIENT 4
VOA-1056
VOA-3993
COSM584
PIK3CA (Chr.3)
COSM328028
TP53* (Chr.17)
COSM99729
PATIENT 6
VOA-3723
VOA-4627
VOA-4698
PATIENT 7
iOvCa250
c.3949G>C (Hm)
c.1156T>C (Ht)
c.1156T>C (Ht)
c.2164G>A (Ht) c.2164G>A (Ht)
c.1416A>T (Ht)
c.1416A>T (Ht)
c.1416A>T (Ht)
c.1416A>T (Ht)
c.1621A>C (Ht)
c.1621A>C (Ht)
c.1621A>C (Ht)
c.35G>T (Ht)
COSM5020653
NRAS* (Chr.1)
PATIENT 5
VOA-3448
c.1124A>G (Ht) c.1124A>G (Ht)
c.182A>G (Ht)
c.182A>G (Ht)
c.1173A>G (Ht) c.1173A>G (Ht) c.1173A>G (Ht) c.1173A>G (Ht)
c.701G>A (Ht)
c.701G>A (Ht)
Note: (*) Means missense mutations reported as oncogenic, and (#) transient primary cell culture. A total of 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes: ABL1, EGFR, GNAS,
KRAS, PTPN11, AKT1, ERBB2, GNAQ, MET, RB1, ALK, ERBB4, HNF1A, MLH1, RET, APC, EZH2, HRAS, MPL, SMAD4, ATM, FBXW7, IDH1, NOTCH1, SMARCB1, BRAF, FGFR1, JAK2, NPM1, SMO, CDH1, FGFR2, JAK3, NRAS, SRC, CDKN2A, FGFR3, IDH2,
PDGFRA, STK11, CSF1R, FLT3, KDR, PIK3CA, TP53, CTNNB1, GNA11, KIT, PTEN, VHL.
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MEK inhibition in low-grade serous cell lines
common oncogenes and tumor suppressor
genes were screened (Table 2). Sanger
sequencing was performed to confirm missense mutations using methods previously
described [25]. Primer sequences used for verification are listed in Supplementary Table 2.
Priming sites for -12 M13 forward and -27 M13
reverse were added to the 5’ ends to allow
direct Sanger sequencing of amplicons [26].
CNV analysis was done using Illumina®
HumanOmni 2.5M-8 Array or CytoScan® HD
array (Affymetrix, Inc) according to manufacture’s protocols. Nexus Copy NumberTM
(BioDiscovery, Inc.) software was used to analyze the copy number data from these two
platforms.
In vitro drug sensitivity assay
Trametinib (GSK1120212; Sellekchem, Cat.
No. S2673), selumetinib (AZD6244; Cat. No.
S1008), binimetinib (MEK162; Cat. No. S7007),
and refametinib (Bay 86-9766; Cat. No. S1089)
were purchased from Selleck Chemicals
(Houston, TX, USA). Dimethylsulfoxide or DMSO
(Sigma, Cat. No. D2650) was purchased from
Sigma-Aldrich (Oakville, Ontario, Canada). LGSC
cells were seeded in 96-well plates at 30-40%
confluence. At 24 hours cells were treated with
DMSO (control) or MEKi (trametinib, selumetinib, binimetinib, or refametinib) using a
range of different drug concentrations.
Inhibitory concentration (IC50, or concentration for 50% of maximal inhibition of cell proliferation) was determined using crystal violet
assay after 72 h drug treatment.
Immunoassays to detect ERK1/2 phosphorylation
Western blot analysis: LGSC cell lysates were
prepared according to previously published protocols [27, 28]. Cell lysates (20 μg) were separated on 8% SDS-PAGE gel and transferred to
nitrocellulose membranes. Primary antibodies
included ERK1/2 (Millipore, Cat. No. 06-182),
p-MAPK (p-ERK1/2, Cell signaling, Cat.No.
4376S), MEK1/2 (Santa Cruz Biotechnologies,
Cat. No. 436), p-MEK1/2 (Cell Signaling, Cat.
No. 9154S), c-PARP (Cell Signaling, Cat. No.
9541S). Vinculin (V9131, Sigma) was used as a
protein loading control. The appropriate horseradish peroxidase (HRP)-conjugated secondary
antibodies (goat-anti-mouse or goat-anti-rabbit,
Sigma Cat. No. A9917 and A0545) were used
accordingly. Western blots were imaged using
Immobilon HRP reagent (Cat. No. WBKLS0500,
2238
Millipore, Etobicoke, ON, Canada) and developed by autoradiograph.
Capillary isoelectric point focusing proteomic
analysis: Native capillary isoelectric point
focusing (cIEF) was performed to assess total
and phosphorylated ERK isoform expression
using NanoPro1000™ System (ProteinSimple™,
Santa Clara, CA) according to manufacturer
protocols [23]. G2 premix gradient (pH 5-8)
(Cat. No. 040-972, ProteinSimple™), ERK 1/2
(Cat. No. 040-474, ProteinSimple™) primary
antibody and Goat anti-Rabbit (Santa Cruz
sc-2054) Human Absorbed IgG secondary antibody was used to identify the protein isoforms.
For both cIEF and Western blot studies, cells
were treated with 20 ng/ml of EGF (Cat. No.
PHG0315, Invitrogen) for 10 minutes.
Cell proliferation assays
Cell proliferation was measured using the
IncuCyte™ (Essen Biosciences, Ann Arbor, MI,
USA). LGSC cell lines were plated at 15-20%
confluence on 96 well plates. After 24 hours,
cells were treated once with DMSO (control) or
MEKi (trametinib 0.1 and 0.5 μM; selumetinib,
binimetinib, and refametinib 1 and 5 μM). The
IncuCyte, a real-time imaging system using a
non-labeled cell monolayer confluence approach, was used to monitor the cell growth by taking phase contrast images of cells at six hour
intervals for 5-6 days. Data analysis was performed using IncuCyte™ cell proliferation assay
software. Results are representative of three
independent experiments, containing five technical replicates.
Cell viability assay
Cell viability was measured using the MTS-Cell
Titer 96R Aqueous Non-Radioactive Cell
Proliferation Assay following the manufacturer’s recommendations (Cat. No. G5430,
Promega, Madison, WI, USA). LGSC cells were
seeded at 15-20% confluence in 96-well plates.
Cells were treated the next day with DMSO control or drug (trametinib -0.1 and 0.5 μM, or selumetinib, binimetinib, and refametinib at 1 and 5
μM) and incubated for 5-6 days. Then, the
media in each well was replaced with 100 μL of
fresh media and 20 μL of MTS reagent. Plates
were incubated for 3.5 hours at 37°C in humidified 5% CO2. Absorbance at 490 nm was measured using microplate reader (BioTek Epoch
SN257811). Cell viability after MEKi treatment
was compared to DMSO treated cells.
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
Figure 1. Graphical representation of copy number variation according to chromosome number. The top graph is a compilation of all cell lines with increased
copy number in blue and decreased in red. For each individual cell line, regions of loss of heterozygosity are represented by a yellow line (below the copy number
changes), whereas a purple line represents regions of allelic imbalance.
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MEK inhibition in low-grade serous cell lines
Apoptosis assay
Apoptosis was measured using a CaspaseGlo® 3/7 Assay Kit (Cat. No. Cat. No. G8090,
Promega, Madison, WI, USA). Briefly, iOvCa241,
VOA-1056 and VOA-4627 LGSC cells (8×103,
5×103, 4×103 cells/96-well for 24 h treatment
and, 6×103, 4×103, 3×103 cells/96-well for 72
h treatment) were incubated for 24 and 72
hours with vehicle (DMSO) or MEKi (trametinib
0.1 μM and 0.5 μM; or selumetinib, binimetinib
or refametinib 1 μM and 5 μM). At the end of
each treatment, luminescence of each well was
measured in a plate-reading luminometer
(Tecan Infinite M200Pro). Experiments were
performed in triplicate for all conditions and
cell lines.
Results
LGSC cell lines have unique cellular and molecular characteristics
Patient clinical data and cell line information
used in this study are summarized in Table 1.
All cell lines were derived from tumor tissues or
ascites from patients with invasive LGSC
(advanced or recurrent), though the original
patient diagnosis varied as expected (borderline, micropapillary borderline and LGSC). A
total of 10 LGSC cell cultures, derived from 7
different patients, were studied and molecularly characterized, though one became senescent (VOA-3667) and another (iOvCa250) grew
too slowly to be used for most experiments.
Three cell line pairs were derived from the
same patient at different time-points in their
disease (VOA 1056;VOA-3993, VOA-3448;VOA3723, and VOA-4627;VOA-4698). Compared to
other ovarian cancer cell lines, LGSC cell lines
(Supplementary Figure 1) show vacuolization,
strong cell adhesion properties (resistance to
trypsinization), and low proliferation rates. STR
analyses were used to define microsatellite loci
for each new cell line and results were compared to the DSMZ STR profile website database (https://www.dsmz.de/services/serviceshuman-and-animal-cell-lines/online-str-analysis.html), which confirmed the uniqueness of
these cell lines.
Hotspot analysis in our LGSC cells revealed
missense mutations in 10 oncogenes: KDR
2240
(4/10 cell lines; 3 patients), PIK3CA (4/10; 2),
KRAS (3/10; 3), KIT (3/10; 2), NRAS (2/10, 1),
MET (2/10; 1), FGFR3 (2/10; 1), JAK3 (2/10; 1),
TP53 (2/10, 1), and APC (1/10; 1). As confirmed
by Sanger sequencing, 1 to 3 missense mutations were detected per case (Table 2). Only 3
of these missense mutations (KRAS, NRAS,
and TP53 genes) are known to be pathogenic
variants according to COSMIC and NCBI ClinVar
classification (Table 2). Mutations in RAS
(KRAS, NRAS) genes were the most frequent
and they were detected in 5 out of 10 cell lines
derived from 4 different patients (Table 1). No
mutations in BRAF were found in the 10 cell
lines, and KRAS and NRAS mutations were
found to be mutually exclusive. Two cell lines
(VOA-4627, VOA-4698), which were derived
from the same patient, carried a TP53 missense mutation. This uncommon finding has
been confirmed histologically and recently
reported as an unusual LGSC of the peritoneum, potentially arising from a low-grade serous tubal intra-epithelial carcinoma (STIC) in a
patient harbouring a BRCA1 mutation [29]. All
missense mutations identified in the paired
samples tested were stable over time.
Subsequent analysis of gene copy-number variation (CNV) was performed in 7 of the cell lines
(derived from 6 patients) as shown in Figure 1.
There were remarkable differences in the percentage of genomic aberrations (2-40%) among
them, but interestingly all cell lines shared copy
number loss affecting the chromosome region
9p21.3. This loss was homozygous in all but
one cell line (VOA-4627). Amplification of chromosomes 8, 12 (containing the KRAS locus)
and 20 were also commonly detected. Copy
number gains in chromosomes 12 and 20 were
detected in 5 out of 7 cell lines. Of note,
chromosomes 4 and 13 were very well conserved. CNV changes were analyzed over time
in one of our paired sample (VOA-1056 and
VOA-3993). In this case, both cell lines shared
similar copy number alteration profiles, however there were significant copy number gains
in chromosomes 2, 12, and 20 in the recurrent
sample (VOA-3993) following treatment with
chemotherapy (Supplementary Figure 2). VOA4627 showed loss of chromosome 1p, a finding
that has been previously observed in LGSC [30]
and also showed high levels of chromosomal
copy number abnormalities and deletions than
the other cell lines (Figure 1). Interestingly, all
LGSC cell lines showed copy number changes
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
Figure 2. Inhibitory Concentration (IC50) values of LGSC cell lines in response to different MEKi treatments. Tra = trametinib, Sel = selumetinib,
Bin = binimetinib, Ref = refametinib. The top graph is an expanded scale
of the bottom graph in order to better show the lower IC50 values. Four
replicates were performed for each treatment condition. All experiments
were performed in triplicate.
(either gain or loss) in common MAPK-pathway
related genes (Supplementary Table 3).
LGSC cell lines that carried oncogenic mutations in KRAS (iOvCa241, VOA-1312) and NRAS
(VOA-1056, VOA-3993) genes were found to
have copy number gain with allelic imbalance in
the same chromosomal regions containing
these loci (chr12:25,357,722-25,403,865 for
KRAS, and chr1:115,247,084-115,259,515 for
NRAS). Furthermore, iOvCa241 showed copy
number gain in BRAF, and VOA-3993 showed
copy number gain in KRAS. In the case of the
unusual cell line carrying mutations in TP53
and BRCA1, allelic imbalance in multiple
regions without clear copy number change was
identified. Copy number changes in relation to
the genes tested for hot spot analysis, as
shown in Supplementary Table 3.
LGSC cell lines have different sensitivities to
MEK inhibitors
Drug sensitivity experiments were conducted
using four different MEKi (trametinib, selumetinib, binimetinib and refametinib), the first
three of which are investigational drugs for the
treatment of advanced/recurrent LGSC patients. We determined the IC50 of each drug in
each LGSC cell line (Figure 2). Figure 2 shows a
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comparison of the IC50 for each
drug, and all of the cell lines tested were most sensitive to trametinib with IC50 values in the nM
range (versus μM for all other
drugs). Next, the same cell lines
were used to study the impact of
all four MEKi’s on proliferation.
Using a live cell-imaging instrument (Incucyte®), we assessed
the effect of a single drug dose
on cell proliferation over a 5 day
period. Based on our IC50 data
and drug dosing from previous
studies [31-33], we chose the following MEKi doses for these
experiments: 0.1 μM trametinib,
and 1 μM selumetinib/binimetinib/refametinib. MEKi treatments
showed the greatest inhibitory
effects on cell lines harbouring
oncogenic mutations in KRAS
(iOvCa241, VOA-1312). In keeping with the IC50 results, trametinib showed the strongest inhibitory effect across all LGSC cell
lines (Figure 3).
Notable differences in proliferation were
observed between cell lines in response to
MEKi treatment (Figure 3). Complete inhibition
of proliferation was observed in iOvCa241
(KRAS G12D) and VOA-1312 (KRAS G12V) in
response to treatment with any MEKi, and most
of the cells remaining on the plate were visibly
non-viable when the images were examined.
Therefore, at the doses tested, drug efficacy
was comparable for all MEKi in these two highly
sensitive cell lines. In contrast, MEKi treatment
in the other cell lines showed less anti-proliferative effects and the observed effects
depended on the specific MEKi used for treatment. In the less sensitive cells (VOA-1056,
VOA-3993, VOA-3448, VOA-3723, VOA-4627,
VOA-4698), trametinib showed the greatest
anti-proliferative activity (Figure 3, patients #3,
#4, #5). Using an alternative assay to measure
cell viability (tetrazolium assay reagent; data
not shown) we confirmed the differential cell
sensitivity and MEKi efficacy results detected
in the proliferation assays (data not shown).
MEK inhibitors vary in their efficacy at blocking
ERK1/2 phosphorylation
After detecting different proliferation and viability effects among the four MEKi’s, we analyzed
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
Figure 3. MEKi effects on LGSC cell proliferation. Single dose (at time 0) MEKi proliferation curves for each cell line
starting at 20% confluence. Proliferation was measured using Incucyte®. Five replicates were performed for each
experimental condition. Experimental results were confirmed by independent experiments performed in duplicate.
the degree of on-target efficacy by measuring
the levels of ERK1/2 phosphorylation
(p-ERK1/2), a downstream effector of MEK. To
2242
analyze p-ERK1/2 we used two protein-based
techniques: Western blot (WB) and capillary
isoelectric point focusing technology (cIEF). As
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
Figure 4. MEKi on-target effects on ERK1/2 phosphorylation in LGSC cells. A: Baseline ERK and p-ERK1/2 by western blot is shown for each cell line. B: ERK and p-ERK1/2 levels in response to MEKi treatment. Tra = trametinib,
Ref = refametinib, Sel = selumetinib, Bin = binimetinib. Films were intentionally overexposed to detect signal in
lanes with low p-ERK1/2 expression. For each cell line all treatment conditions were performed as part of the same
experiment. All experimental findings were confirmed in independent experiments performed in triplicate. Note:
membranes incubated with p-ERK1/2 antibody were overexposed to capture all remaining signal after MEKi treatment. WB images have been cut to keep the same sample order. All membranes incubated with the same antibody
have been exposed for the same period of time.
seen in Figure 4A, basal levels of p-ERK1/2
varied among cell lines, being lower in
iOvCa241, VOA-1056, VOA-3448, VOA-4627,
and higher in iOvCa250, VOA-1312, VOA-3723,
VOA-3993, and VOA-4698. Of note, iOvCa241
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cells only expressed the ERK1 isoform (ERK2
homozygous copy loss was detected in the CNV
array), and VOA-1056 cells predominantly
expressed ERK2. Interestingly, an increase in
p-ERK1/2 was detected in all three of the recur-
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
Figure 5. Time course and dose-response effects of MEKi treatment in LGSC cell lines. A: WB expression levels of
ERK1/2, MEK and PARP after MEKi treatments with different dosing (low versus high) over time (24 h versus 72
h). B: Apoptosis induction measured by caspase 3/7 after MEKi treatment in LGSC cell lines. All experiments were
repeated. Note: Caspase 3/7 control measurements at 24 h and 72 h were subtracted from all treatment values
in the graphs shown.
rent cell line samples compared to their patientmatched initial cell line samples (VOA-1056 vs
VOA-993; VOA-3448 vs. VOA-3723; VOA-4627
vs. VOA-4698). This increase in p-ERK1/2 was
observed over time independently of patient
treatment.
We then treated each cell line with the different
MEKi (trametinib 0.1 μM, selumetinib 1 μM,
binimetinib 1 μM, or refametinib 1 μM), and
analyzed the levels of p-ERK1/2 expression at
24 hours. For most cell lines, 1 μM selumetinib
or binimetinib only partially inhibited p-ERK1/2,
there was greater inhibition using 1 μM refametinib, and complete inhibition with 0.1 μM
trametinib (Figure 4B). EGF treatment was used
to determine if each MEKi could still suppress
phosphorylation after MAPK pathway stimulation. Only trametinib was able to completely
block the induction of p-ERK1/2. To further
2244
characterize drug effect on p-ERK1/2 isoforms
on native protein lysates, we selected 4 cell
lines for further study using cIEF: the sensitive
iOvCa241 cell line, and one cell line from each
of the paired samples (VOA-1056, VOA 3723,
and VOA-4627). The results from these experiments are shown in Supplementary Figure 3.
LGSC cells treated with 0.1 μM trametinib
showed little detectable levels of p-ERK1/2
across cell lines. Refametinib (1 μM) was the
second most effective inhibitor of p-ERK1/2.
Using cIEF we could not observe any drug-related differences in p-ERK1/2 in the treated
sensitive cell line (iOvCa241) as treatment with
any of the MEKi resulted in complete inhibition
of p-ERK1/2 signal. Only partial inhibition of
p-ERK1/2 was observed after treatment of the
resistant cell lines using 1 μM selumetinib or
binimetinib. As observed by WB, iOvCa241 cells
only expressed ERK1, and VOA-1056 cells predominantly expressed ERK2.
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
Figure 6. Anti-proliferative effects of MEKi treatment (low and high dose) in LGSC cells. Proliferation experiments
showing the effects of low dose (1×) and high dose (5×) of MEKi treatment (trametinib, selumetinib, binimetinib,
refametinib) in three different LGSC cell lines. (*) P-value >0.5 comparing high dose versus low dose for each drug
in the iOvCa241 cell line. (§) P-value <0.05 comparing high dose versus low dose for each drug, except trametinib
(p-value = 0.09), in the VOA-1056 cell line. (¶) P-value <0.05 comparing high dose versus low dose for each drug in
the VOA-4627 cell line.
MEK inhibitor anti-proliferative effects correlate with MEKi on-target efficacy over time
To further explore the inhibitory effects of MEKi
treatment in LGSC cells, we performed doseresponse experiments on the 4 selected cell
lines. Each cell line was treated with low (0.1 or
1 μM) and high (0.5 or 5 μM) doses of each
MEKi for periods of 24 and 72 hours. p-ERK1/2
expression levels were measured as a marker
for MEK activity. In the sensitive iOvCa241, all
drugs completely inhibited p-ERK1/2 at any
dose or time (Figure 5A). In the other 3 cell
lines (VOA-1056, VOA-3723, and VOA-4627)
there were similarities in drug-dependent sup2245
pression of p-ERK1/2 levels. Trametinib completely blocked p-ERK1/2 even at low dose (0.1
μM) over 72 hours, however selumetinib or
binimetinib at high dose (5 μM) failed to completely inhibit p-ERK1/2. In contrast, refametinib showed complete inhibition of p-ERK1/2
when used at 5 μM over 72 hours. In general,
MEKi treatment resulted in an increase in
MEK1/2 phosphorylation (p-MEK1/2). Interestingly, in the sensitive iOvCa241 line, no clear
increase was observed after trametinib
treatment.
We then examined the effects of high-dose
MEKi treatment on cell proliferation and
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
p-ERK1/2 expression. In iOvCa241, higher dosing (Figure 6) did not further reduce proliferation rates, as low doses completely inhibited
proliferation (P>0.5; t-test, high vs. low dose of
each drug; 120 h ours post-treatment). Notably,
in the 3 resistant cell lines (VOA-1056, VOA3723, and VOA-4627) there were drug-dependent differences in proliferation after higher
drug dosing. For example, in the most resistant
cell line, VOA-4627, each MEKi significantly
decreased proliferation rates 120 hours posttreatment (P<0.05; t-test, high dose vs. low
dose of each drug) (Supplementary Table 4). In
contrast, for VOA-1056, statistically significant
decreases in proliferation were observed at
120 hours post-treatment for all drugs (P<0.05;
t-test) except trametinib (P = 0.09; t-test, high
dose vs. low dose). In 3 more resistant cell
lines, dose dependent reductions in p-ERK1/2
over time (Figure 5A) correlated with degree of
anti-proliferative effect for all drugs except
trametinib (Figure 6). Once again, trametinib
and refametinib more effectively blocked
p-ERK1/2 and show the greatest anti-proliferative activity across all tested LGSC cell lines.
MEKi treatment induces apoptosis and the
degree of apoptosis correlates with drug sensitivity
Using the same low and high doses of each
MEKi, we then analyzed the levels of PARP activation (cleaved PARP or c-PARP) in four LGSC
cell lines by WB (Figure 5A). Three cell lines
(iOvCa241, VOA-1056 and VOA-3723) had little
or no detectable baseline c-PARP activity, but
considerable c-PARP induction after MEKi
treatment. In contrast, VOA-4627 showed considerable baseline PARP activity without a corresponding increase after MEKi treatment. As
another marker for apoptosis activation, we
also measured the level of caspase 3/7 cleavage in 3 cell lines (iOVCa241, VOA-1056, and
VOA-4627). Figure 5B shows the changes in
cleaved caspase 3/7 (above substracted baseline control) pre-treatment and 24 and 72 hours
post-treatment. All MEKi increased cleaved
caspase levels at 24 and 72 hours. The MEKi
sensitive iOvCa241 cell line showed the most
robust apoptotic response to MEKi treatment
and trametinib at low dose was as effective at
inducing apoptosis as the high dose of any
other MEKi (Figure 5B). The MEKi resistant
VOA-4627 cell line showed no difference in
caspase activity when treated with lower doses
2246
of MEKi over time (P = 0.08; t-test: 24 vs. 72
hours for all MEKi). In contrast, the less resistant VOA-1056 cells, showed significant increases in cleaved caspase after 72 h treatment
(P<0.00001; t-test 24 vs. 72 hours, for all
MEKi).
Discussion
We have established and molecularly characterized a cohort of advanced/recurrent LGSC
cell lines. LGSC cells are difficult to culture and
only a few LGSC cell lines (MPSC-1, HOC-7) are
available and studied to date [24, 34, 35].
Clinical and molecular characterization (pathology at collection, tumor stage, treatment
status, STR profiling, mutation and CNV analyses) of cell lines provides a basis for using
these lines as experimental models. For
example, previous tumor mutational studies
have shown that BRAF mutations are found in
LGSC, but are mostly confined to early-stage
cases [30, 36, 37]. As such cases are often
cured by surgery, the use of BRAF mutant LGSC
cell lines for investigational drug studies will be
of limited value. No BRAF mutations were identified in our cell line cohort in agreement with a
previous study [38], though there has been one
reported patient with a BRAF fusion who
achieved a complete response to binimetinib in
combination with paclitaxel [39]. KRAS/NRAS
mutations are relatively frequent in advanced/
recurrent LGSC [16], and in our study they were
detected in 5 out of 10 cell lines from 4 different patients. Further study of LGSC cell lines
with KRAS/NRAS mutations is very important
to understand how these mutations affect
pathway signalling and responses to MAPK
pathway inhibition in LGSC. Successful targeted therapies against KRAS mutations have
proven to be challenging [40, 41] and it is not
clear whether MEKi will effectively treat LGSC
patients with KRAS mutations [23].
Cancer hotspot mutational analysis revealed
that cell lines derived from tumor samples
obtained from the same patient at different
times during the course of the disease shared
the same mutations (see mutations in cell line
pairs VOA-1056 and VOA-3993; VOA-3348 and
VOA-3723; and VOA-4627 and VOA-4698;
Table 2). Copy number analysis showed marked
heterogeneity among LGSC cell lines. Further
genomic disruption was seen over time, or in
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
response to treatment, in the paired cell lines
that we analyzed, VOA-1056 and VOA-3993.
Despite this heterogeneity, LGSC cell lines
shared common regions of copy number
change. Loss of chromosome 9p was detected
in all cell lines, an event that has been observed
in other recent genomic profiling studies done
in LGSC tumors [42-44]. This genomic region is
known to contain the CDKN2A/B locus, which
encodes for three well-known tumor suppressor genes: p14 (Arf), p15 (INK4b), and p16
(INK4a). Loss of p16 has been described in
LGSC tumors in comparison to their high-grade
counterparts [2, 45]. It is of interest that loss of
chromosome 9 is frequently seen in LGSC but
is rare in SBOT, suggesting a potential role for
malignant transformation [42, 43]. Other copy
number changes were observed among cell
lines included gains in chromosomes 8, 12 and
20. Gains in chromosome 12 have been
described in both LGSC and SBOT [43, 46].
Notably, KRAS is located on chromosome 8.
More in-depth study of these regions may help
further characterize LGSC in patients with
relapsed or progressive disease.
We used our LGSC cell lines to test four MEKi
and noted remarkable differences in the sensitivity of these lines to the individual drugs tested. We noted that the proliferation assays were
helpful in terms of classifying sensitive versus
resistant cells as the results from the IC50
experiments did not well characterized the efficacy of the different drugs over time. Based on
patterns of growth inhibition and apoptosis, we
identified two sensitive cell lines (VOA-1312
and iOvCa241). These two lines demonstrated
marked inhibition of proliferation in response to
all MEKi tested even at low doses. The
iOvCa241, MEKi sensitive cells, also showed a
marked apoptotic response after MEKi treatment when compared to two MEKi resistant
cell lines (VOA-1056 and VOA-4627) (Figure 5).
The remaining cell lines show varying degrees
of resistance to MEKi with less anti-proliferative effects in response to drug. In most MEKi
resistant cell lines, though a dose-response
effect on proliferation was observed, proliferation continued despite higher doses of drug
(See Figure 3). To confirm the significance of
these findings it will be important to test additional LGSC cell lines derived from patients
known to be sensitive or resistant to MEKi
treatment.
2247
Our evaluation of the biological effects of four
different MEKi in LGSC cells showed that following a single treatment, trametinib is a more
effective inhibitor of cell proliferation at 10-fold
lower doses than the other drugs tested (refametinib, selumetinib, and binimetinib). Trametinib
also showed the greatest reduction in cell proliferation and viability in the more resistant cell
lines. These observations are similar to recent
reports of MEKi testing in lung cancer cell lines
and tumor models [32, 47]. Trametinib most
effectively induced apoptosis in our cell lines,
which has been described as an important
mechanism of MEKi action [31].
The study of on-target effects of each MEKi in
our LGSC cell lines revealed interesting differences relating to cellular p-ERK1/2 at baseline
and in response to drug treatment. In our study,
there was no correlation between baseline
p-ERK levels and MEKi sensitivity in keeping
with a previous report [33]. We did observe a
remarkable correlation between the degree of
inhibition of p-ERK1/2 levels and the anti-proliferative effects of each MEKi. Interestingly, we
observed that binimetinib, as well as selumetinib, was much less effective at inhibiting
p-ERK1/2 and cell proliferation in LGSC cell
lines than trametinib or refametinib. Dose
dependent inhibition of p-ERK1/2 was
observed in our study for all drugs except trametinib, which effectively blocked p-ERK1/2
even at low doses (0.1 μM). This finding is in
keeping with a study on breast cancer cell lines
showing no correlation between trametinib
IC50 values and inhibition of ERK signaling as
p-ERK1/2 is inhibited at trametinib concentrations not affecting proliferation [33]. Our results
therefore suggest that using a particular MEKi
dosing schedule or drug that fails to eliminate
p-ERK1/2 will not result in maximal anti-proliferative effects in tumors from LGSC patients.
On the other hand, cells with intrinsic resistance to MEKi continue to proliferate despite
marked inhibition of p-ERK1/2. This observation was most apparent using trametinib as it
so effectively inhibits ERK phosphorylation at
low concentrations. Recently, an investigational drug trial using binimetinib in LGSC was
closed following an interim analysis that failed
to show a pre-defined benefit on progressionfree survival compared to chemotherapy. A
large randomized trial is currently open
(NCT02101788) to evaluate trametinib as a
Am J Cancer Res 2016;6(10):2235-2251
MEK inhibition in low-grade serous cell lines
treatment for LGSC and this will be a very
informative trial relating to the use of MEKi in
this disease.
In 2012, an important phase 2 clinical trial was
reported evaluating selumetinib as a treatment
for recurrent LGSC. There was a 15% response
rate and KRAS/BRAF mutations were not a predictor of response. Copy number changes or
other MAPK-pathway related aberations were
not evaluated. As clinical responses to selumetinib were infrequent, it will be important to
identify biomarkers of MEKi response in order
to treat those patients who are most likely to
benefit. In the present study, the most sensitive
cell lines (VOA-1312 and iOvCa241) were found
to have mutations in KRAS (G12V and G12D
respectively) with associated gene copy-number gains. This finding suggests that these lines
may be highly dependent on KRAS activity. Polh
et al. found that primary ovarian cancer cell
lines and cultures with BRAF (4 cell lines) and
KRAS mutations (2 cell lines) were more sensitive to MEKi (CI-1040) than 6 wild type lines
[24]. In a study of colon cancer cell lines, MEKi
treatment impaired anchorage-independent
growth of nearly all KRAS/BRAF mutants, but
not wild type cells. However, neither increased
baseline p-ERK1/2 nor a reduction of p-ERK1/2
secondary to MEKi treatment correlated with
inhibition of anchorage independent growth
[48]. Recently, it has been suggested that
KRAS mutations may in fact result in a hyperexcitable KRAS rather than an altered protein
in a statically active state [41]. MAPK signaling
is very complex as there are copy number changes, other pathway mutations, and cross talk
mechansims all of which can affect MAPK signaling and the response to MEKi drug
treatment.
This study shows marked differences in LGSC
cell line MEKi efficacy. We observed that trametinib is highly effective in blocking p-ERK1/2
at concentrations ten-fold lower than the other
MEKi, and this on-target effect is reflected in its
in vitro drug efficacy. As effective systemic
treatments are lacking for this disease and no
xenograft models for pre-clinical testing are
available, these cell lines should be used to
better understand drug- or tumor-related determinants of MEKi sensitivity. There is a need for
this work as investigational MEKi (trametinib
and pimasertib) are currently being tested in
large randomized trials designed for drug regis2248
tration and approval. These trials are proceeding with a very limited understanding of MAPK
pathway function in LGSC. With recent advances in molecular medicine, pre-clinical investigational drug testing can play an important role
in the design of clinical trials, molecular companion studies, and the selection of the most
promising investigational drugs for clinical testing. Linking molecular research strategies with
drug development and clinical trial design will
be necessary in order to improve treatment
outcomes in rare cancer types such as LGSC.
Acknowledgements
This work was supported by the British
Columbia Cancer Foundation, the OvCaRe
Program, and the London Run for Ovarian
Cancer. We would also like to thank Janine Senz
for all of her technical support and Megan
Froats for her graph design expertise. The authors extend a special thanks to the MacKenzie
family, and to all the patients, families, and
donors for who have supported this research
study.
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Mark S Carey,
Department of Obstetrics & Gynaecology, University
of British Columbia, Vancouver, British Columbia,
Canada. E-mail: [email protected]
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